Chemical and Structural Analysis of Eucalyptus Globulus and E
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ORIGINAL RESEARCH ARTICLE published: 16 September 2014 doi: 10.3389/fpls.2014.00481 Chemical and structural analysis of Eucalyptus globulus and E. camaldulensis leaf cuticles: a lipidized cell wall region Paula Guzmán1*, Victoria Fernández1*, José Graça 2 , Vanessa Cabral 2 , Nour Kayali 3 , Mohamed Khayet 4 and Luis Gil 1 1 Forest Genetics and Ecophysiology Research Group, School of Forest Engineering, Technical University of Madrid, Madrid, Spain 2 Instituto Superior de Agronomia, Centro de Estudos Florestais, Universidade de Lisboa, Lisboa, Portugal 3 Mass Spectrometry Unit, Faculty of Chemistry, University Complutense of Madrid, Madrid, Spain 4 Department of Applied Physics I, Faculty of Physics, University Complutense of Madrid, Madrid, Spain Edited by: The plant cuticle has traditionally been conceived as an independent hydrophobic layer Anja Geitmann, Université de that covers the external epidermal cell wall. Due to its complexity, the existing relationship Montréal, Canada between cuticle chemical composition and ultra-structure remains unclear to date. This Reviewed by: study aimed to examine the link between chemical composition and structure of isolated, Mary Lai Preuss, Webster University, USA adaxial leaf cuticles of Eucalyptus camaldulensis and E. globulus by the gradual extraction Heiner Goldbach, University of Bonn, and identification of lipid constituents (cutin and soluble lipids), coupled to spectroscopic Germany and microscopic analyses. The soluble compounds and cutin monomers identified could *Correspondence: not be assigned to a concrete internal cuticle ultra-structure. After cutin depolymerization, a Victoria Fernández and Paula Guzmán, cellulose network resembling the cell wall was observed, with different structural patterns Forest Genetics and Ecophysiology Research Group, School of Forest in the regions ascribed to the cuticle proper and cuticular layer, respectively. Our results Engineering, Technical University of suggest that the current cuticle model should be revised, stressing the presence and Madrid, Ciudad Universitaria s/n, major role of cell wall polysaccharides. It is concluded that the cuticle may be interpreted Madrid 28040, Spain e-mail: [email protected]; as a modified cell wall region which contains additional lipids. The major heterogeneity of [email protected] the plant cuticle makes it difficult to establish a direct link between cuticle chemistry and structure with the existing methodologies. Keywords: cell wall, cuticle, cutin, eucalypt, leaf, lipids, polysaccharides, ultra-structure INTRODUCTION alcohols, alkanes, aldehydes, esters and ketones) and aromatic The cuticle is an asymmetric, composite layer covering the sur- compounds (Jetter et al., 2006). Polysaccharides present in the face of most aerial organs of plants (Tyree et al., 1990; Fernández cuticle are similar to those found in the cell wall (i.e., cellulose, and Brown, 2013), which is mainly extruded into the external hemicelluloses and pectins; López-Casado et al., 2007; Guzmán wall of epidermal cells (Pollard et al., 2008; Javelle et al., 2010). et al., 2014b). Based on the relative abundance of chemical con- Due to its location at the interface between the plant and the stituents (Schmidt and Schönherr, 1982; Heredia, 2003; Samuels surrounding environment, the cuticle plays an array of crucial et al., 2008), the cuticle has been conceived as a hydrophobic protective and physiological functions (Remus-Emsermann et al., membrane. 2011; Serrano et al., 2014). Additionally, it has a role in organ The cuticle has been generally described as having three dif- growth and differentiation (Kutschera and Niklas, 2007; Takada ferent layers from the external to the internal surface, i.e.,: (i) and Iida, 2014). the epicuticular wax (EW) layer, (ii) the cuticle proper (CP), and The multi-functional character of the plant cuticle is pro- (iii) the cuticular layer (CL; von Mohl, 1847; Jeffree, 2006). It has vided by its complex and heterogeneous chemical and physical been traditionally assumed that cutin/cutan and waxes are found nature (Gouret et al., 1993; Guzmán et al., 2014a). From a chem- both in the CP and CL, while the presence of polysaccharides is ical viewpoint, the cuticle is generally defined as a cutin and/or restricted to the CL (von Mohl, 1847; Jeffree, 2006). By contrast, cutan polymer matrix, with epicuticular waxes deposited on to the presence of cellulose and pectins in both cuticular layers has its outer surface, embedded intracuticular waxes and phenolics, been recently reported (Guzmán et al., 2014b). For various species and polysaccharides stemming from the cell wall (Domínguez and organs, different patterns of cuticular ultra-structure have et al., 2011). Cutin is formed by a network of inter-esterified, been categorized on the basis of transmission electron microscopy hydroxy and hydroxy-epoxy C16 and/or C18 fatty acids (Kolat- (TEM) observations (Holloway, 1982; Jeffree, 2006). However, a tukudy, 1980), sometimes linked with glycerol (Graça et al., 2002). fair degree of cuticular structural variation may arise from e.g., The chemical composition of an alternative cuticle matrix poly- the TEM sample preparation procedure implemented as recently mer named cutan remains controversial (Tegelaar et al., 1991; shown by Guzmán et al. (2014a). Deshmukh et al., 2005). Cuticular waxes (soluble cuticular lipids) The cuticular chemical composition and/or ultra-structure of are complex mixtures of aliphatic (mainly long-chain fatty acids, a few organs and species has been studied by gradual extraction www.frontiersin.org September 2014 | Volume 5 | Article 481 | 1 Guzmán et al. The cuticle: a modified cell wall and analysis of different cuticular fractions (e.g., Fernández et al., temperature (23–25◦C) for 1 month, manually shaking the flasks 2011, 2014; Tsubaki et al., 2013; Belge et al., 2014), and using at frequent time intervals. After the extraction period, clean intact diverse microscopic means (e.g., Canet et al., 1996; Casado and adaxial cuticles were selected, thoroughly washed in deionized Heredia, 2001; Koch et al., 2004). The potential link between water, air-dried, and stored for further use. chemistry and structure of different cuticular fractions or spe- cific molecular constituents has been examined chiefly focusing on EXTRACTION AND DETERMINATION OF SOLUBLE COMPOUNDS EW (e.g., Baker, 1982; Barthlott et al., 1998; Jeffree, 2006). Inte- Isolated cuticles were cut into small pieces, wrapped in filter grative studies on the internal cuticle are however, scarce (e.g., paper and successively extracted in 150 mL of dichloromethane Wattendorff and Holloway, 1982; Gouret et al., 1993; Viougeas (6 h; Sigma–Aldrich), ethanol (12 h; Sigma–Aldrich) and distilled et al., 1995; Yeats et al., 2012), and the existing relationship water (24 h) using a Soxhlet apparatus. Solvents were subsequently between chemical composition and structure remains unclear so evaporated to dryness with a rotary evaporator and the material far (Khayet and Fernández, 2012). In addition, there is currently a extracted was weighed and stored. Soxhlet extracted (SE) cuticles limited understanding of the potential cross-links and/or molecu- were also stored for further analysis. lar assemblies taking place between cuticular chemical constituents The cuticular material extracted in dichloromethane was ana- (Pollard et al., 2008; Kunst and Samuels, 2009). lyzed by gas chromatography-mass spectrometry (GC-MS). Sam- Given the chemical and physical complexity of the plant ples were filtered using polytetrafluoroethylene (PTFE) filters prior cuticle, there is a need for carrying out comprehensive investi- to injection in a GC-MS apparatus (GCVarian CP-3800 coupled to gations which may help us understand the cuticle in a holistic a MS ion trap Varian Saturn 2200, GC-ITMS, automatic injector manner. The high complexity of the cuticle together with an COMBI-PAL; CTC Analytics, Zwingen, Switzerland). The chro- array of technical constrains may lead to theoretical miscon- matographic conditions were as follows (86 min per run): injection ceptions concerning the structure, chemistry and function of volume 1 μL, with Helium as carrier gas (1.2 mL min−1), and the cuticle (Guzmán et al., 2014a,b). In this study we aimed at injector temperature 290◦C. The column (SLB-5 ms fused silica analyzing the link between ultra-structure and chemical com- capillary column, 30 m × 0.25 mm × 0.25 μm film thickness; position (focusing on lipids) of the leaf cuticle, following an Supelco, Sigma–Aldrich) was set to 40◦C (3 min), heating rate integrative approach. For this purpose Eucalyptus camaldulen- of 10◦C min−1 up to 290◦C (18 min), and 20◦C min−1 up to sis and E. globulus were used as model species due to their 310◦C (13 min). The MS conditions were: 70 eV ionization volt- major importance for forestry production (Guzmán et al., 2013). age, 150◦C trap temperature, 20–550 units of mass scan range Furthermore, despite such species are native to different habi- with 8 min solvent delay. The compounds were identified and tats their taxonomic proximity may involve a certain degree of quantified by comparing their mass spectra with NIST library cuticular similarity. By merging different analytical and electron spectra and analyzing the corresponding peaks of GC-MS ion microscopy techniques before and after the selective extraction chromatograms. and identification of chemical constituents, the following ques- tions were addressed: (i) is the adaxial leaf cuticle of both species